In shotgun proteomics, the gold standard technique is reversed-phase liquid chromatography coupledto mass spectrometry. Many researches have been carried out to study the effects on identification per ... [more ▼]

In shotgun proteomics, the gold standard technique is reversed-phase liquid chromatography coupledto mass spectrometry. Many researches have been carried out to study the effects on identification per-formances of chromatographic parameters such as the stationary phase and column dimensions, mobilephase composition and flow rate, as well as the gradient slope and length. However, little attention isusually paid to the injection solvent composition.In this study, we investigated the effect of the injection solvent on protein identification parameters(number of distinct peptides, amino acid coverage and MS/MS search score) as well as sensitivity. Trypticpeptides from six different proteins, covering a wide range of physicochemical properties, were employedas training set. Design of experiments was employed as a tool to highlight the factors related to thecomposition of the injection solvent that significantly influenced the obtained results. Optimal resultsfor the training set were applied to analysis of more complex samples. The experiments pointed outoptimising the composition of the injection solvent had a strong beneficial effect on all the consideredresponses. On the basis of these results, an approach to determine optimal conditions was proposed tomaximise the protein identification performances and detection sensitivity. [less ▲]

BACKGROUND: Mitral regurgitation is a frequent valvular heart disease affecting around 2.5 % of the population with prevalence directly related to aging. Degeneration of mitral valve is broadly considered ... [more ▼]

BACKGROUND: Mitral regurgitation is a frequent valvular heart disease affecting around 2.5 % of the population with prevalence directly related to aging. Degeneration of mitral valve is broadly considered as a passive ongoing pathophysiological process and little is known about its physiological deregulation. The purpose of this study was to highlight new biomarkers of mitral regurgitation in order to decipher the underlying pathological mechanism as well as to allow the diagnosis and the monitoring of the disease. RESULTS: Modulation of various blood proteins expression was examined in patients suffering from different grades of mitral regurgitation (mild, moderate and severe) compared to healthy controls. To this end, several routine clinical assays and the multi analyte profile technology targeting 184 proteins were used. High-density lipoprotein, apolipoprotein-A1, haptoglobin and haptoglobin-alpha2 chain levels significantly decreased proportionally to the degree of mitral regurgitation when compared to controls. High-density lipoprotein and apolipoprotein-A1 levels were associated with effective regurgitant orifice area and regurgitant volume. Apolipoprotein-A1 was an independent predictor of severe mitral regurgitation. Moreover, with ordinal logistic regression, apolipoprotein-A1 remained the only independent factor associated with mitral regurgitation. In addition, myxomatous mitral valves were studied by immunocytochemistry. We observed an increase of LC3, the marker of autophagy, in myxomatous mitral valves compared with healthy mitral valves. CONCLUSION: These potential biomarkers of mitral regurgitation highlighted different cellular processes that could be modified in myxomatous degenerescence: reverse cholesterol transport, antioxidant properties and autophagy. [less ▲]

BACKGROUND: Dried blood analysis experiences a growing interest due to practical, ethical and financial advantages compared with classical wet plasma or serum analysis. Besides classical DBS, new alternatives are commercialized as volumetric absorptive microsampling (VAMS) that are expected to overcome hematocrit influence. RESULTS: The feasibility of hepcidin (a peptide hormone) extraction and determination from DBS and VAMS blood sampling was investigated. Experimental design was used to determine the optimal extraction conditions. Matrix effect and extraction recovery were studied and a special attention was paid to phospholipid removal. CONCLUSION: The data suggest that the combination of VAMS and phospholipid removal plates provides low matrix effect and high sensitivity, and constitutes an easy and promising protocol for hepcidin analysis. [less ▲]

Acute graft-versus-host disease (aGVHD) remains a life-threatening complication of hematopoietic stem cell transplantation (HSCT), limiting its application. To optimize management of aGVHD and reduce therapy-related toxicity, early specific markers are needed. The main objective of this study was thus to uncover diagnostic biomarkers comparing plasma protein profiles of patients at the time of acute GVHD diagnosis and of patients undergoing HSCT without aGVHD. Additional analysis of samples taken 15 days before aGVHD diagnosis was also performed to evaluate the potential of the newly discovered biomarkers for early diagnosis. To extract a maximum of information from plasma samples, we used three complementary proteomic approaches, namely 2D-DIGE, SELDI-TOF-MS and 2D-LC-MSE. We identified and confirmed by means of a independent techniques, the differential expression of several proteins indicating significantly increased inflammation response and disturbance in the coagulation cascade. The variation of these proteins was already observed 15 days before GVHD diagnosis, suggesting the potential early detection of the disease before symptoms appearance. Finally, logistic regression analysis determines a composite biomarker panel comprising fibrinogen, fragment of fibrinogen beta chain, SAA, prothrombin fragments, apolipoprotein A1 and hepcidin that optimally discriminated patients with and without GVHD. The area under the receiver operating characteristic curve distinguishing these 2 groups was 0.95. [less ▲]

To date, precise roles of EMD remain poorly described. In this paper, we investigate the role of EMD in the C16-ceramide autophagy pathway. Ceramides are bioactive signalling molecules acting notably in ... [more ▼]

To date, precise roles of EMD remain poorly described. In this paper, we investigate the role of EMD in the C16-ceramide autophagy pathway. Ceramides are bioactive signalling molecules acting notably in the regulation of cell growth, differentiation or cell death. However, the mechanisms by which they mediate these pathways are not fully understood. We found that C16-ceramide induces EMD phosphorylation on its LEM domain through PRKACA. Upon ceramide treatment, phosphorylated EMD binds LC3 leading to an increase of the autophagosomes formation. These data suggest a new role of EMD as an enhancer of autophagosomes formation in the C16-ceramide autophagy pathway in colon cancer cells. [less ▲]

Angina is chest pain induced by ischemia of the heart muscle, generally due to obstruction or spasm of the coronary arteries. People that suffer from average to severe cases of angina have an increased ... [more ▼]

Angina is chest pain induced by ischemia of the heart muscle, generally due to obstruction or spasm of the coronary arteries. People that suffer from average to severe cases of angina have an increased percentage of death before the age of 55, usually around 60%. Therefore, prevention of major complications, optimizing diagnosis, prognosis and therapeutics are of primary importance. The main objective of this study was to uncover biomarkers by comparing serum protein profiles of patients suffering from stable or unstable angina and controls. We identified by non-targeted proteomic approach and confirmed by the means of independent techniques, the differential expression of several proteins indicating significantly increased vascular inflammation response, disturbance in the lipid metabolism and in atherogenic plaques stability. [less ▲]

Background: We analysed kinetics of IL-7 and IL-15 levels in 70 patients given peripheral blood stem cells after nonmyeloablative conditioning. Methods: EDTA-anticoagulated plasma and serum samples were obtained before conditioning and about once per week after transplantation until day 100. Samples were aliquoted and stored at 280uC within 3 hours after collection until measurement of cytokines. IL-7 and IL-15 levels were measured by ELISAs. Results: Median IL-7 plasma levels remained below 6 pg/L throughout the first 100 days, although IL-7 plasma levels were significantly higher on days 7 (5.1 pg/mL, P = 0.002), 14 (5.2 pg/mL, P,0.001), and 28 (5.1 pg/mL, P = 0.03) (but not thereafter) than before transplantation (median value of 3.8 pg/mL). Median IL-15 serum levels were significantly higher on days 7 (12.5 pg/mL, P,0.001), 14 (10.5 pg/mL, P,0.001), and 28 (6.2 pg/mL, P,0.001) than before transplantation (median value of 2.4 pg/mL). Importantly, IL-7 and IL-15 levels on days 7 or 14 after transplantation did not predict grade II–IV acute GVHD. Conclusions: These data suggest that IL-7 and IL-15 levels remain relatively low after nonmyeloablative transplantation, and that IL-7 and IL-15 levels early after nonmyeloablative transplantation do not predict for acute GVHD. [less ▲]

OBJECTIVE: Knee osteoarthritis (OA) is a heterogeneous, complex joint pathology of unknown aetiology. Biomarkers have been widely used to investigate OA but currently available biomarkers lack specificity ... [more ▼]

OBJECTIVE: Knee osteoarthritis (OA) is a heterogeneous, complex joint pathology of unknown aetiology. Biomarkers have been widely used to investigate OA but currently available biomarkers lack specificity and sensitivity. Therefore, novel biomarkers are needed to better understand the pathophysiological processes of OA initiation and progression. METHODS: Surface enhanced laser desorption/ionisation-time of flight-mass spectrometry proteomic technique was used to analyse protein expression levels in 284 serum samples from patients with knee OA classified according to Kellgren and Lawrence (K&L) score (0-4). OA serum samples were also compared to serum samples provided by healthy individuals (negative control subjects; NC; n=36) and rheumatoid arthritis (RA) patients (n=25). Proteins that gave similar signal in all K&L groups of OA patients were ignored, whereas proteins with increased or decreased levels of expression were selected for further studies. RESULTS: Two proteins were found to be expressed at higher levels in sera of OA patients at all four K&L scores compared to NC and RA, and were identified as V65 vitronectin fragment and C3fpeptide. Of the two remaining proteins, one showed increased expression (unknown protein at m/z of 3762) and the other (identified as connective tissue-activating peptide III protein) was decreased in K&L scores >2 subsets compared to NC, RA and K&L scores 0 or 1 subsets. CONCLUSION: The authors detected four unexpected biomarkers (V65 vitronectin fragment, C3f peptide, CTAP-III and m/z 3762 protein) that could be relevant in the pathophysiological process of OA as having significant correlation with parameters reflecting local inflammation and bone remodelling, as well as decrease in cartilage turnover. [less ▲]

Introduction: Cardiac troponin I and T are specific markers of myocardial injury that are widely used for the diagnosis of acute coronary syndrome (ACS). In acute chest pain without ST-segment elevation ... [more ▼]

Introduction: Cardiac troponin I and T are specific markers of myocardial injury that are widely used for the diagnosis of acute coronary syndrome (ACS). In acute chest pain without ST-segment elevation, they are used to differentiate unstable angina from non ST-segment elevation myocardial infarction (NSTEMI). Recently, troponin assays with higher analytical sensitivities became available to enable the detection of minor myocardial damage and identify individuals at higher risk for ACS. As a result of its high tissue-specificity, cardiac troponin T and I are cardio-specific, highly sensitive markers for myocardial damage. The aim of this study was to evaluate the new higher sensitive troponin (T and I) in patients with stable angina and acute chest pain without ST-segment elevation. Methods: Sixty subjects (mean age : 65.5± 11 years), were included: 20 healthy controls, 20 patients with stable angina, 9 with unstable angina (troponin-) and 18 patients with NSTEMI myocardial infarction (troponin+). The protocol was approved by the ethic committee of the University of Liège (Belgium). High sensitive troponin T (hsTnT) determination was realized on heparin plasma by electrochemiluminescence immunoassay on Modular E (Roche Diagnostic). Troponin I II (TnI II) is a chemiluminescent microparticle immunoassay for the quantitative determination of cardiac troponin-I in heparine plasma on the ARCHITECT i System (Abbott Diagnostic). The lower detection limit of these assays was 0.005μg/L for hsTnT and 0.01μg/L for TnI II. Stastistical analysis was performed using t test. P value <0.05 was considered significant. Results: HsTNT levels were 0.003(0.003, 0.004) [median baseline (1st, 3rd quartile)]ng/ml in controls, 0.0075 (0.00475, 0.014) ng/ml in stable angina, 0.011(0.006, 0.012) ng/ml in unstable angina and 0.3715 (0.1795, 1.00725) ng/ml in NSTEMI ACS. TnI II levels were 0 (0, 0.001) ng/ml in controls and in patients with stable angina, 0.07 (0.005, 0.014) ng/ml in unstable angina and 1.4475 (0.0407, 2.656) ng/ml in NSTEMI. HsTNT and TnI II levels were significantly increased in NSTEMI as compared to control subjects, patients with stable and unstable angina. TnI II levels were also increased in unstable angina as compared to controls. Conclusion: In our population, TnI II was more sensitive than hsTNT to detect minor myocardial damage in patients with unstable angina as compared to controls. Therefore, future studies will have to determine whether TnI II might contribute to better risk stratification and treatment strategy in this group of patients. [less ▲]

The oncogenic protein BCL-3 activates or represses gene transcription through binding with the NF-kappaB proteins p50 and p52 and is degraded through a phospho- and GSK3-dependent pathway. However, the ... [more ▼]

The oncogenic protein BCL-3 activates or represses gene transcription through binding with the NF-kappaB proteins p50 and p52 and is degraded through a phospho- and GSK3-dependent pathway. However, the mechanisms underlying its degradation remain poorly understood. Yeast-two-hybrid analysis led to the identification of the proteasome subunit PSMB1 as a BCL-3-associated protein. The binding of BCL-3 to PSMB1 is required for its degradation through the proteasome. Indeed, PSMB1-depleted cells are defective in degrading polyubiquitinated BCL-3. The N-terminal part of BCL-3 includes lysines 13 and 26 required for the K48-linked polyubiquitination of BCL-3. Moreover, the E3 ligase FBW7 known to polyubiquitinate a variety of substrates phosphorylated by GSK3 is dispensable for BCL-3 degradation. Thus, our data defined an unique motif of BCL-3 that is needed for its recruitment to the proteasome and identified PSMB1 as a key protein required for the proteasome-mediated degradation of a nuclear and oncogenic IkappaB protein. [less ▲]

In most diseases, the clinical need for serum/plasma markers has never been so crucial, not only for diagnosis, but also for the selection of the most efficient therapies, as well as exclusion of ... [more ▼]

In most diseases, the clinical need for serum/plasma markers has never been so crucial, not only for diagnosis, but also for the selection of the most efficient therapies, as well as exclusion of ineffective or toxic treatment. Due to the high sample complexity, prefractionation is essential for exploring the deep proteome and finding specific markers. In this study, three different sample preparation methods (i.e., highly abundant protein precipitation, restricted access materials (RAM) combined with IMAC chromatography and peptide ligand affinity beads) were investigated in order to select the best fractionation step for further differential proteomic experiments focusing on the LMW proteome (MW inferior to 40,000 Da). Indeed, the aim was not to cover the entire plasma/serum proteome, but to enrich potentially interesting tissue leakage proteins. These three methods were evaluated on their reproducibility, on the SELDI-TOF-MS peptide/protein peaks generated after fractionation and on the information supplied. The studied methods appeared to give complementary information and presented good reproducibility (below 20%). Peptide ligand affinity beads were found to provide efficient depletion of HMW proteins and peak enrichment in protein/peptide profiles. [less ▲]

Constitutive nuclear factor (NF)-kappaB activation in haematological malignancies is caused in several cases by loss of function mutations within the coding sequence of NF-kappaB inhibitory molecules such ... [more ▼]

Constitutive nuclear factor (NF)-kappaB activation in haematological malignancies is caused in several cases by loss of function mutations within the coding sequence of NF-kappaB inhibitory molecules such as IkappaBalpha or p100. Hut-78, a truncated form of p100, constitutively generates p52 and contributes to the development of T-cell lymphomas but the molecular mechanism underlying this oncogenic potential remains unclear. We show here that MMP9 gene expression is induced through the alternative NF-kappaB-activating pathway in fibroblasts and also on Hut-78 or p52 overexpression in fibroblasts as well as in lymphoma cells. p52 is critical for Hut-78-mediated MMP9 gene induction as a Hut-78 mutant as well as other truncated NF-kappaB2 proteins that are not processed into p52 failed to induce the expression of this metalloproteinase. Conversely, MMP9 gene expression is impaired in p52-depleted HUT-78 cells. Interestingly, MLL1 and MLL2 H3K4 methyltransferase complexes are tethered by p52 on the MMP9 but not on the IkappaBalpha promoter, and the H3K4 trimethyltransferase activity recruited on the MMP9 promoter is impaired in p52-depleted HUT-78 cells. Moreover, MLL1 and MLL2 are associated with Hut-78 in a native chromatin-enriched extract. Thus, we identified a molecular mechanism by which the recruitment of a H3K4 histone methyltransferase complex on the promoter of a NF-kappaB-dependent gene induces its expression and potentially the invasive potential of lymphoma cells harbouring constitutive activity of the alternative NF-kappaB-activating pathway. [less ▲]

Asthma is a complex inflammatory disease of airways. A network of reciprocal interactions between inflammatory cells, peptidic mediators, extracellular matrix components, and proteases is thought to be ... [more ▼]

Asthma is a complex inflammatory disease of airways. A network of reciprocal interactions between inflammatory cells, peptidic mediators, extracellular matrix components, and proteases is thought to be involved in the installation and maintenance of asthma-related airway inflammation and remodeling. To date, new proteic mediators displaying significant activity in the pathophysiology of asthma are still to be unveiled. The main objective of this study was to uncover potential target proteins by using surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) on lung samples from mouse models of allergen-induced airway inflammation and remodeling. In this model, we pointed out several protein or peptide peaks that were preferentially expressed in diseased mice as compared to controls. We report the identification of different five proteins: found inflammatory zone 1 or RELM (FIZZ-1), calcyclin (S100A6), clara cell secretory protein 10 (CC10), Ubiquitin, and Histone H4. [less ▲]